Methods for moving or manipulating living cells are essential tools that enable research directed to therapies relating to stem cells, in vitro fertilization, cell and tissue culture, tissue regeneration and similar fields. For example, widely used stem cells, whether derived from embryos (ES), induced pluripotent stem cells (IPS), cord blood cells, adult cells such as skin or other tissues, have great promise for therapeutic action because of the ability of these pluripotent cells to differentiate into different terminal cell types. While this ability has enabled recent developments in tissue replacement therapy, manipulating stem cell colonies can be very time consuming and difficult. Cell manipulations that enable propagation of stem cells—in particular, isolating cells or cell culture colonies for transport (“passaging”)—remain highly laborious and technically demanding (see, e.g., Cooke, J. A. and Minger, S. L. Culture of Human Stem Cells Ch. 2, 2007, incorporated herein by reference).
Fewer methods exist for moving cells maintained in culture, as confluent layers (“sheets”) or otherwise adherent to the surface of a tissue culture dish pose difficult problems if these cells need to be moved or manipulated. Stem cells in particular exist in colonies grown on substrates surrounded by nutrient-rich growth medium, frequently in Petri dishes. Normal growth of these cells in culture requires passaging—division of cells in to another chamber containing fresh growth medium. For some cell types, it may be further desirable to isolate a small population of cells from a larger population, or a portion of a layer, and transfer those cells to a different location for further testing, study or treatment.
Maintaining undifferentiated stem cells in long term culture presents a special problem, however. Stem cell colonies tend to differentiate over time in vitro. Therefore to maintain the stem cell line, or to significantly multiply the number of cells, the undifferentiated portions (or selected colonies) must be isolated and passaged to new containers. Ideally, excision and passaging should be done without compromising the colony, that is, without killing large numbers of cells. Although cell passaging using enzymes like trypsin or collagenase to release adherent cells from their substrate has been in routine use for decades, use of enzymes in stem cell cultures is particularly disfavored because of the increased risk of genetic alterations.
Non-enzymatic, mechanical methods have been described for achieving the goal of excising or cutting cells from the substrate on which they are grown. For example, it is well known in the art that cells can be excised from a confluent layer by making scoring or cutting the layer with a finely drawn glass micropipette. After cutting, the layer “pieces”—small clusters of cells—can be lifted off of the surface and placed in a different environment. Manual cutting using drawn glass micropipettes is preferred because this method minimizes the chance of genetic alteration of stem cells.
Another mechanical method for separating cells involves the use of piezoelectric microknives, for example, the MicroChisel Piezo-Power Microdissection (PPMD) system (see, e.g., Harsch, M. A. et al. Am. J. Pathol. 158:1985-90, 2001). PPMD employs a sharpened tungsten needle as a microscopic knife that oscillates from small piezoelectric vibrations to dissect cells from surrounding tissue. While simple, this method lacks precision and accuracy on a cellular level, and undoubtedly kills or damages cells in large numbers due to lateral vibrations and contact with the micropipette or knife.
While PPMD may be a suitable technique in certain circumstances, in other applications it will be particularly important to avoid mechanical damage. This is especially true in cases where a relatively few number of high value cells must be isolated and cannot be lost to excessive tissue damage. In other cases, cell damage or destruction may release harmful chemicals into the tissue culture medium that could lead to an adverse impact on cell growth, physiology, or function. Another disadvantage to these physical methods for cell manipulation is that cutting devices can introduce foreign materials into the cell colony, possibly contaminating or otherwise compromising the integrity of the culture.
Lasers present an alternative means for the micromanipulation of living cells and tissue. The art has disclosed infrared laser “traps,” sometimes referred to as “optical tweezers,” that use forces of radiation pressure to manipulate entire living cells or organelles within cells (see, e.g., Ashkin, A. and Dziedzic, J. M. Nature 330:769-71, 1987). Lasers have also been used to “weld” detached retinas, to sculpt the cornea to achieve different optical focusing in myopic patients, and to eliminate unwanted cells in a culture.
Lasers have been also useful for cutting biological material such as fixed cells in tissue samples. “Laser scissors” have been developed where cells (or a portion thereof) can be literally cut by a laser beam acting as a scalpel. Lasers can be used to create micron-sized pores in cell membranes in a process called optoporation. These small pores seal quickly and do not result in permanent damage, but while open, the pore might permit chemicals to enter the inside of the cell that are normally excluded by the cell membrane, including large molecules like DNA that, if introduced, might change the genetic makeup of the cell. Laser manipulation has also proven useful for the assisted hatching of human eggs in fertility assistance programs and clinics. Laser ablation, for example, using the ZILOS-tk (λ=1450 nm), has been used in vitro to thin or even remove a small area of the zona pellucida surrounding embryos; this procedure is used to enhance implantation of the new embryo without damaging it, and to enable a portion of the embryo trophectoderm to emerge and be removed for trophectoderm pre-implantation genetic diagnosis (see, e.g., Pangalos, C. G. et al. Fetal Diagn. Ther. 24:334-339, 2008).
Lasers avoid many problems associated with the enzymatic or mechanical methods for cell manipulation described above. There is no contamination since the laser cutting beam is non-material and cannot introduce any foreign material into the growth chamber. Microscope-based laser systems using an automatic stage can be set up to divide specimens into small areas with micron precision for subsequent passaging, in patterns that can be re-run automatically as necessary.
However, because of the high amounts of energy contained in a laser beam, one problem in laser manipulation is heat generation and damage to adjacent structures. Lasers used in cell manipulation are often operated in short sub-microsecond pulses to deliver brief doses of energy. The overall energy to the system can be finely tuned by changing one or more parameters such as the laser power, the duration of the pulse, and the number of pulses. Local heating will increase as the power and the overall pulse duration increase.
According to International Standard IEC 60825-1 Amendment 2 (see also 21C.F.R. §1040.10), which is accepted by the Food and Drug Administration (see, e.g., FDA Laser Notice No. 50), lasers are classified by their ability to cause biological damage to the eye or skin during use. Based on laser wavelength, beam power, and pulse duration, classifications proceed from Class I, safe under all conditions of normal use, to Class IV, high power lasers that can burn the skin or ignite combustible materials, in addition to causing potentially devastating and permanent eye damage. Class I lasers are generally believed to have insufficient power for cutting or burning.
A laser microdissection system called PALM® uses a UV N2 laser (λ=337 nm, 3 ns pulse duration, with peak pulse power in excess of 10 kW) to provide a system for the retrieval of selected cell populations and single cells from tissue sections (see, e.g., Vogel, A. et al. Meth. Cell Biol. Vol. 82, Ch. 5, 2007). A frequency-tripled Nd:YAG laser at 355 nm has also been used for this purpose. Pulses from the UV laser are focused through the microscope to cause laser ablation of cells and tissue in a tissue section. The sample is generally not under a medium as it is necessary to minimize the effective sample mass. PALM is thought to operate through a photochemical mechanism that breaks down biological material into atoms that are blown away from the sample at supersonic velocities. This cutting action is restricted to a tiny focal spot of the laser (<1 μm), leaving adjacent material like neighboring cells or nearby nucleic acids and proteins intact (see, e.g., Schutze, K. et al. Cell. Mol. Biol. (Noisy-le-grand) 44:735-746, 1998). A second pulse propels the cut-out sample into a collection device.
Despite these achievements, the use of UV photons carries the risk of actinic effects, and the Class III or Class IV lasers required to produce the extremely short and intense pulses required to cut biological materials are potentially hazardous and costly to operate.
Therefore, the need exists in the art to provide a reliable, cost-effective Class I laser-based system and method for safely manipulating cells (e.g., cutting cells, excising sheets of cells for further examination or manipulation, etc.).